Congo red analogues as potential anti-prion agents

Article abstract of DOI:10.1016/S0014-827X(03)00151-4

'Transmissible Spongiform Encephalopathies' (TSE) are a group of degenerative, progressive and fatal disorders of CNS which affect both humans and animals, characterised by a long incubation time. The pathogenetic mechanism in TSE is the conversion of normal prion protein (PrP^sen) to an altered protease resistant isoform (PrP^res) that accumulates in amyloid deposits into the brain; therefore, PrP^res is the primary target for therapeutic strategies. The discovery that the sulphonated azo dye Congo red (CR) is able to inhibit the replications of TSE agents and the accumulation of PrP^res in animals and in scrapie infected mouse neuroblastoma cells induced us to designe molecules structurally related to CR (1a-f, 2f,g). The compounds were tested in vitro to evaluate their interaction with 263K PrP^res. Six of the tested compounds were found to interact with PrP^res molecules and to over-stabilise the PrP^res aggregates, as CR does. However, none of them induced the reversion of PrP ^res to PrP^sen.

Full text of DOI:10.1016/S0014-827X(03)00151-4

Il Farmaco 58 (2003) 929ꢀ937
/
www.elsevier.com/locate/farmac
Congo red analogues as potential anti-prion agents
Stefania Villa a,*, Giorgio Cignarella a, Daniela Barlocco a, Marco Gervasoni b,
Gabriella Carcassola c, Laura Giannino c, Paolo Mantegazza b
a Istituto di Chimica Farmaceutica e Tossicologica, Universita` degli Studi di Milano, Viale Abruzzi 42, I-20131 Milan, Italy
b Dipartimento di Farmacologia, Chemioterapia e Tossicologia Medica, Universita` degli Studi di Milano, Via Vanvitelli 32, I-20129 Milan, Italy
c Dipartimento di Patologia Animale, Igiene e Sanita` Pubblica Veterinaria, Universita` degli Studi di Milano, Via Celoria 10, I-20133 Milan, Italy
Received 13 December 2002; accepted 28 January 2003
Abstract
‘Transmissible Spongiform Encephalopathies’ (TSE) are a group of degenerative, progressive and fatal disorders of CNS which
affect both humans and animals, characterised by a long incubation time. The pathogenetic mechanism in TSE is the conversion of
normal prion protein (PrPsen) to an altered protease resistant isoform (PrPres) that accumulates in amyloid deposits into the brain;
therefore, PrPres is the primary target for therapeutic strategies. The discovery that the sulphonated azo dye Congo red (CR) is able
to inhibit the replications of TSE agents and the accumulation of PrPres in animals and in scrapie infected mouse neuroblastoma
cells induced us to designe molecules structurally related to CR (1aꢀf, 2f,g). The compounds were tested in vitro to evaluate their
/
interaction with 263K PrPres. Six of the tested compounds were found to interact with PrPres molecules and to over-stabilise the
PrPres aggregates, as CR does. However, none of them induced the reversion of PrPres to PrPsen
.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Congo red; Transmissible spongiform encephalopathies (TSE); Anti-prion-molecules
1. Introduction
The pathogenic mechanism of the disease is the
conversion of normal prion protein (PrPsen) to a
protease resistant isoform (PrPres) that accumulates in
Transmissible Spongiform Encephalopathies (TSE)
are fatal neurodegenerative disorders in animals and
humans. They are characterized by a long incubation
period and a slow progression. TSE are represented
amyloid deposits into the brain [5ꢀ
7]; therefore, PrPres is
/
the primary target for therapeutic strategies. Since
neither control tools nor prophilaxis are currently
available for viral or bacterial infections, the necessity
for therapeutic agents is highly desiderable.
principally by Creutzfeldtꢀ
/Jakob disease (CJD),
GerstmannꢀStrausslerꢀScheinker Syndrome (GSS)
/
/
¨
The recent discovery that the azoic dye Congo red
(CR) [8] is able to inhibit the deposition of PrP in
amyloid plaques [9,10] induced us to project and
evaluate new molecules structurally related to CR (see
Table 1). We assumed that CR was metabolised by
endogenus azareductase to release 1a that could be the
real active agent, in analogy with other azoic prodrugs
(e.g. Sulphasalazine [11]). So we considered as reference
compound 3,4-diamino-1-naphthalen-1-sulphonic acid
(1b) as the sodium salt (1a) formally derived from CR
by hydrogenolysis. However, 1a was unexpectedly found
unstable not only in aqueous solution but also as such
thus making the in vitro test unreliable. Therefore, the
corresponding acid 1b was regarded as the parent
compound. In addition 1c, 1d in which the sulphonic
and Fatal Familial Insomnia (FFI) in humans and by
scrapie and Bovine Spongiform Encephalopathy in
animals (BSE).
The BSE epizootic recently spread in UK and the first
cases of a new human spongiform encephalopathy,
called ‘new juvenile variant CJD’ (vCJD) [1,2], alarmed
the entire scientific community for the risk of bovine
prion transmission to human. This hypothesis was also
supported by the discovery that vCJD is caused by the
same prion found in BSE [3,4].
* Corresponding author.
E-mail address: stefania.villa@unimi.it (S. Villa).
´
0014-827X/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0014-827X(03)00151-4

930
S. Villa et al. / Il Farmaco 58 (2003) 929ꢀ937
/
Table 1
acetylation to 8 which was nitrated to give a mixture
of 2-nitro (10a) and 4-nitro (10b) derivatives which were
separated by flash chromatography. Deacetylation of
10a with 6 N HCl followed by catalytic reduction gave
2f isolated as the hydrochloride (see Scheme 4). Chlor-
osulphonation of 8 followed by deacetylation with 6 N
HCl led to 2g (see Scheme 5). Attempts to synthesise the
5,6,7,8-tetrahydro analogue 2b of 1b by nitration of 2g
followed by catalytic reduction, failed (Fig. 1).
Compounds R1
R2
R3
Over-stabilising PrP aggregates
(WB)
3. Biology
All compounds were tested in a simple cell-free system
composed of partially purified PrPres, to evaluate their
ability in over-stabilising the PrPres and in inducing the
reversion of the protease resistant PrPres into the normal
protease sensitive PrPsen. CR was used as the reference
compound [9].
RC
ꢂ
/
ꢂ
/
ꢂ
/
/
ꢂ
/
1b
1c
1d
1e
1f
NH2 NH2 SO3H
NH2 NH2 COOH
NH2 NH2 SO2NH2
ꢂ
/
ꢂ
/
ꢂ
ꢂ
/
ꢂ
/
ꢂ
/
ꢂ
/
NH2
H
SO3Na
H
-
NH2 NH2
NH2 NH2
ꢂ
/
ꢂ
/
2f
2g
H
SO3H
ꢂ
/
ꢂ
/
NH2
H
-
4. Experimental
group of 1b was, respectively replaced by a carboxyl or
sulphonamide group, and 1f which missed the acidic
function were prepared. Finally to verify the importance
of the naphthalenic moiety, the tetralin analogues of 1f
(2f) and 1g (2g) were synthesized. Unfortunately, any
attempt to obtain the partially reduced analogue of 1b
failed.
4.1. Chemistry
Melting points (8C) were determined on a Buchi 510
¨
capillary melting point apparatus and are uncorrected.
Analyses indicated by the symbols were within 90.4 of
/
1
the theoretical values. H NMR spectra were recorded
on a Brucker AC200 spectrometer; chemical shift are
reported as d (ppm) relative to tetramethylsilane (TMS)
as internal standard. Dimethyl-d6 sulphoxide was used
as the solvent, unless otherwise noted. The TLC on silica
gel plates was used to check product purity. Silica gel 60
2. Chemistry
The 3,4-diamino-naphthalen-1-sulphonic-acid (1b)
was synthesised from CR by reduction with stannous
chloride in concentrated hydrochloric acid according to
a reported method [12]. The corresponding sodium salt
1a still unreported in literature, was prepared by
reduction of CR with hydrazine monohydrate in the
presence of catalytic amount of 10% Pd/C in ethanol
(see Scheme 1). 3,4-Diamino-naphthalen-1-carboxylic
acid (1c) was obtained from the known 3-nitro-naphtha-
len-1-carboxylic acid [13] by reaction with hydroxyla-
mine hydrochloride in the presence of potassium
hydroxide (3) [14] followed by catalytic (10% Pd/C)
reduction (see Scheme 3). Analogously, 1f (1,2-diamino-
naphthalene) was obtained from the commercially
available 2-nitro-naphthalene (see Scheme 3). The
sulphonamide 1d was synthesised from CR as the acetyl
derivative 4 by reaction with POCl3 and PCl5 followed
by treatment of the thus obtained 5 with 30% NH4OH
to give 6 which was eventually reduced to 1d as above
described for 1a (see Schemes 1 and 2). The compound
2f was synthesised starting from the commercially
available 5,6,7,8-tetrahydro-1-amino-naphthalene by
(Merck 70ꢀ230 Mesh) was used for flash column
/
chromatography. The structure of all compounds were
consistent with their analytical and spectroscopic data.
The compounds CR and 1e are commercially available
(Sigmaꢀ/Aldrich) and were used without further purifi-
cation.
4.1.1. 3,4-Diamino-naphthalene-1-sulphonic acid sodium
salt (1a)
To a solution of CR (1 g, 1.4 mmol) in EtOH (20 ml)
hydrazine hydrate (1 ml, 21 mmol) was added dropwise
and the mixture was refluxed for 20 h in the presence of
10% Pd/C (10/1 w/w). After filtration of the catalyst and
evaporation in vacuo of the solvent, the residue was
triturated in EtOH (2ꢁ
which was crystallized
C10H9N2NaO3S (260.24) 1H NMR: 8.63 (d, 1H, HÃ
Ar), 7.89 (d, 1H, HÃAr), 7.57 (s, 1H, H-3), 7.05ꢀ7.38
(m, 2H, HÃAr), 5.0 (br s, 4H, 2NH2 exchangeable with
D2O).
/20 ml) to give a yellow solid
from EtOH (76%)
/
/
/
/

S. Villa et al. / Il Farmaco 58 (2003) 929ꢀ
/937
931
Scheme 1.
4.1.2. 3,4-Diamino-naphthalene-1-sulphonic acid (1b)
A solution of stannous chloride (10 g, 52.7 mmol) in
concentrated HCl (37.5 ml) was added dropwise to a
with D2O), 9.0ꢀ
changeable with D2O), 8.8 (s, 1H, H-2), 8.6ꢀ
/
9.2 (m, 3H, 1HÃ
/
Arꢂ
/
2H NH2 ex-
/8.7 (m,
1H, HÃAr), 7.6ꢀ7.9 (m, 2H, HÃAr).
/
/
/
solution of CR (5 g, 7.18 mmol) in waterꢀEtOH (100 ml
/
2:1). The mixture was stirred on a warm water bath until
decolorization and then further refluxed for 2.5 h. After
cooling to 5 8C the yellow solid which separated was
filtered and vigorously shaken with MeOH (10 ml). The
residue was filtered and crystallized from water to give
4.1.4. 3,4-Diamino-naphthalene-1-carboxylic-acid (1c)
A mixture of 3 (0.84 g, 3.62 mmol) and 10% Pdꢀ
(0.17 g) in absolute EtOH (100 ml) was hydrogenated at
r.t. The catalyst was filtered off and the solution was
acidified with HCl in Et2O and evaporated to give 1c
/C
1b (2 g, 59%) C10H10N2O3S (238.17) m.p. 274ꢀ
1H NMR: 8.76 (m, 1H, HÃ
Ar), 8.12 (m, 1H, HÃ
7.85 (s, 1H, H-3), 7.49 (m, 2H, HÃAr).
/
276 8C
(0.82 g, 95%). C11H10N2O2×
1H NMR: 10.2ꢀ
11.0 (br s, 1H, COOH exchangeable
with D2O), 9.1ꢀ9.2 (m, 1H, HÃAr), 8.35ꢀ8.4 (m, 1H,
HÃAr), 8.3 (s, 1H, H-2), 7.4ꢀ7.8 (m, 2H, HÃAr).
/
HCl (238.67) m.p. ꢀ280 8C.
/
/
/
Ar),
/
/
/
/
/
/
/
/
4.1.3. 3-Nitro-4-amino-naphthalene-1-carboxylic acid
(3)
To a solution of 3-nitro-naphthalene-1-carboxylic-
acid (0.94 g, 4.3 mmol) and hydroxylamine hydrochlo-
ride (1.87 g, 27 mmol) in absolute EtOH (45 ml) a
solution of potassium hydroxide (5.61 g, 99.8 mmol) in
absolute EtOH (35 ml) was added dropwise. After
4.1.5. 3,3?-{[1,1?-Biphenyl]-4,4?-diylbis(azo)}bis(4-
acetyl-amino)-naphthalene-1-sulphonic acid sodium salt
(4)
To a mixture of CR (2 g, 2.9 mmol) in AcOH (20 ml)
acetic anhydride (0.57 ml, 5.8 mmol) was added drop-
wise. The solution was refluxed for 1 h. After cooling,
the separated solid was filtered and washed with acetone
heating at 50ꢀ60 8C for 1 h and cooling at room
/
temperature (r.t.) the reaction mixture was poured into
ice and the pH was adjusted to pH 6 with 6 N HCl. The
insoluble was filtered and washed with water to give 3
(2ꢁ
M.p. ꢀ
8.8ꢀ8.9 (m, 2H, HÃ
2H, H-2), 8.1ꢀ8.2 (m, 4H, HÃ
/
5 ml) to yield 4 (2.26 g in almost quantitative yield).
1
/
300 8C C36H26N6Na2O6S2 (780.73) H NMR:
Ar), 8.5ꢀ8.6 (m, 2H, HÃAr), 8.3 (s,
benzidine), 8.0ꢀ8.1 (m,
(0.44 g, 44.5%). M.p. 202ꢀ
/
204 8C C11H8N2O4 (232.19)
/
/
/
/
1H NMR: 12.8ꢀ
/13.2 (br s, 1H, COOH exchangeable
/
/
/

932
S. Villa et al. / Il Farmaco 58 (2003) 929ꢀ937
/
Scheme 2.
4H, HÃ
/
benzidine), 7.5ꢀ
/
7.6 (m, 4H, HÃ
/
Ar), 1.95 (s, 6H,
8.52 (br s, 4H, SO2NH2); 8.49 (d, 2H, HÃ
2H, H-2); 8.18 (d, 4H, HÃbenzidine); 8.02 (d, 4H, HÃ
benzidine); 7.8 (br s, 4H, NH2 exchangeable with D2O);
7.59ꢀ7.8 (m, 4H, HÃAr).
/
Ar); 8.36 (s,
COCH3).
/
/
/
/
4.1.6. 3,3?-{[1,1?-Biphenyl]-4,4?-dilbis(azo)-}bis(4-
amino)-naphthalene-1-sulphonamide (6)
A mixture of 4 (2.0 g, 2.6 mmol), phosphorus
pentachloride (2.0 g, 9.6 mmol) and phosphorus oxy-
chloride (3 ml, 16 mmol) was refluxed for 1 h. After
4.1.7. 3,4-Diamino-naphthalene-1-sulphonamide (1d)
Compound 1d was prepared by hydrogenolysis start-
ing from 6 as above reported for 1a (35%) m.p. 172ꢀ
/
1
cooling, the residue was treated with CH2Cl2 (2ꢁ20 ml)
/
173 8C C10H11N3O2S (237.28) H NMR: 8.43 (d, 1H,
HÃAr): 8.06 (d, 1H, HÃAr); 7.74 (s, 1H, H-2); 7.35ꢀ7.40
and evaporated to dryness. The black solid isolated (2 g)
constituted by crude 5 was directly dissolved in EtOH
(12 ml) and to the solution 30% ammonium hydroxide
(1.5 ml) was slowly added on cooling until pH 10. The
resulting red solution was refluxed for 1.5 h. After
cooling, the insoluble red solid was filtered and tritu-
rated with potassium bicarbonate (10 ml) and then with
/
/
/
(m, 2H, H-6, H-7); 7.14 (s, 2H exchangeable with D2O
SO2NH2); 5.61 (s, 2H exchangeable with D2O NH2);
5.00 (s, 2H exchangeable with D2O NH2).
4.1.8. 1,2-Diamino-naphthalene hydrochloride (1f)
Compound 1f was prepared from the commercial
available 2-nitro-napthalene by reaction with NH2OH
acetone (10 ml) to give 6 (1.16 g, 62%) m.p. ꢀ
C32H26N8O4S2 (650.72). H NMR: 8.84 (d, 2H, HÃAr);
/
300 8C
1
/

S. Villa et al. / Il Farmaco 58 (2003) 929ꢀ
/937
933
followed by catalytic (10% Pd/C) reduction of the thus
obtained 7, as above reported for 1c.
For 7 (45.8%) m.p. 144 8C C10H8N2O2 (188.18) H
2.90ꢀ
/
2.95 (m, 2H, H-5, H-8); 2.7ꢀ
/
2.85 (m, 2H, H-5, H-
8); 2.18 (s, 3H CH3); 1.78ꢀ1.96 (m, 4H, 2H-6, 2H-7).
/
1
NMR: 8.13 (d, 1H, HÃ
1H, HÃAr); 7.62ꢀ7.80 (m, 2H, H-6, H-7); 7.38 (s, 2H
Ar).
For 1f (72%) m.p. 267 8C C10H12Cl2N2 (231.11) H
NMR: 8.56 (s, 6H exchangeable with D2O NH2HCl);
/
Ar); 7.95 (d, 1H, HÃ
/
Ar); 7.79 (d,
4.1.11. 2-Nitro-1-amino-5,6,7,8-tetrahydro-naphthalene
(11)
A solution of 10a (0.5 g, 2.1 mmol) in 6 N HCl (25 ml)
was refluxed for 2 h. After cooling, the pH of the
mixture was adjusted to 10 with 5 N NaOH then
/
/
exchangeable with D2O NH2); 7.10 (d, 1H, HÃ
/
1
8.15 (d, 1H, HÃ
/
Ar); 7.94 (d, 1H, HÃ
/
Ar); 7.48ꢀ7.60 (m,
/
extracted with CH2Cl2 (3ꢁ7 ml). The organic layers
were dried over Na2SO4, filtered and evaporated in
vacuo to give 11 (0.40 g, in almost quantitative yield).
/
2H, H-6, H-7); 7.36 (dd, 2H, H-5, H-8).
M.p. 195ꢀ
(CDCl3): 7.84 (d, 1H, HÃ
4.18 (br s, 2H exchangeable with D2O NH2); 2.9ꢀ
2H, H-5, H-8); 2.43ꢀ2.55 (m, 2H, H-5, H-8); 1.78ꢀ
(m, 4H, 2H-6, 2H-7).
/
197 8C C10H12N2O2 (192.21) 1H NMR
4.1.9. 1-Acetylamino 5,6,7,8-tetrahydro-naphthalene (8)
Compound 8 was obtained by acetylation of the
corresponding amine as above reported for 4 (87%)
/
Ar); 6.70 (d, 1H, HÃ
3.0 (m,
1.96
/
Ar);
/
152 8C C12H15NO (189.26). 1H NMR: 7.52 (d,
Ar, H-exchangeable with
/
/
m.p. 151ꢀ
2H, HÃAr); 7.00 (m, 2H, HÃ
D2O NHCOCH3); 2.76ꢀ2.85 (m, 2H, H-5, H-8); 2.57ꢀ
2.65 (m, 2H, H-5, H-8); 2.18 (s, 3H, CH3); 1.78ꢀ1.96 (m,
4H, 2H-6, 2H-7).
/
/
/
/
/
/
4.1.12. 1,2-Diamino-5,6,7,8-tetrahydro-naphthalene (2f)
Compound 2f was prepared by catalytic hydrogena-
tion starting from 11 as above reported for 1c (86%)
m.p. 234ꢀ
(CDCl3): 9.0ꢀ
H-4); 6.52 (d, 1H, H-3); 2.70ꢀ
1.62ꢀ1.93 (m, 4H, 2H-6, 2H-7).
/
235 8C C10H16N2Cl2 (235.04). 1H NMR
10.0 (br s, 6H, 2NH2HCl); 7.26 (d, H,
3.01 (m, 4H, 2H-5, 2H-8);
4.1.10. 2-Nitro-1-acetylamino-5,6,7,8-tetrahydro-
naphthalene (10a)
To a solution of 8 (10 g, 53 mmol) in AcOH (75 ml)
cooled at 5 8C, fuming HNO3 (9 ml) was added
dropwise. After heating at 40 8C for 3 h the reaction
mixture was cooled at r.t., poured into ice and extracted
/
/
/
4.1.13. 1-Acetylamino-4-solphonylchloride-5,6,7,8-
tetrahydro-naphthalene (9)
with EtOAc (3ꢁ20 ml). The organic layers were dried
/
over Na2SO4, filtered and evaporated in vacuo to give a
mixture of 10a and 10b which were separated by silica
gel flash chromatography eluting with 1:1 petroleum
To cooled chloro-sulphonic acid (3.5 ml, 53 mmol) 8
(2 g, 10.56 mmol) was added with caution keeping the
temperature at 10ꢀ15 8C. After heating at 60 8C for 2 h,
/
etherꢀ
/
EtOAc. 10a (2 g, 16%) m.p. 162ꢀ
/
164 8C
then cooling at r.t. the reaction mixture was poured onto
ice and the insoluble was filtered off and repeatedly
1
C12H14N2O3 (234.26). H NMR: 9.38 (s, 1H exchange-
able with D2O NHCOCH3); 7.80 (dd, 2H, H-3, H-4);
washed with water to give 9 (2.3 g 76.6%). M.p. 184ꢀ
/
Scheme 3.

934
S. Villa et al. / Il Farmaco 58 (2003) 929ꢀ937
/
Scheme 4.
1
187 8C C12H14ClNO3S (287.76). H NMR: 9.25 (s, 1H
exchangeable with D2O NHCOCH3); 7.6 (d, 1H, HÃ
Ar); 7.2 (d, 1H, HÃAr); 2.90ꢀ3.00 (m, 2H, H-5, H-8);
2.43ꢀ2.59 (m, 2H, H-5, H-8); 2.18 (s, 3H CH3); 1.68ꢀ
8); 2.6ꢀ
2H-7).
/
2.8 (m, 2H, H-5, H-8); 1.60ꢀ2.00 (m, 4H, 2H-6,
/
/
/
/
/
/
4.2. Pharmacology
1.95 (m, 4H, 2H-6, 2H-7).
4.2.1. Purification of PrPres
PrPres was partially purified from brains of Syrian
hamster euthanized in the terminal stage after infection
with 263k scrapie strain, as previously described [15];
500 mg of brain was homogenized in 4.5 ml of lysis
buffer (100 mmol NaCl, 10 mmol EDTA, 0.5% NP4O,
4.1.14. 1-Amino-4-sulphonic acid-5,6,7,8-tetrahydro-
naphthalene (2g)
A solution of 9 (2 g, 6.9 mmol) in 6 N HCl (20 ml) was
refluxed for 1 h. After cooling the precipitate was
collected by filtration, washed with water and dried in
0.5% NaDOC, 10 mmol TrisꢀHCl pH 4) and centri-
/
vacuo to give 2g (1.23 g, 68%). M.p. 201ꢀ
/
203 8C
fuged at 6,700 rpm for 15 min at 4 8C. The supernatant
was supplemented with an equal volume of sarcosyl 20%
and centrifuged at 14.000 rpm for 45 min at 4 8C. Tris-
buffered saline containing 0.1% N-tetradecyl-N,N-di-
1
C10H14ClNO3S (262.61) H NMR: 8.8ꢀ
/
10.0 (br s, 4H
exchangeable with D2O NH2HCl, SO3H); 7.7 (d, 1H,
HÃAr); 7.15 (d, 1H, HÃAr); 3.10ꢀ3.3 (m, 2H, H-5, H-
/
/
/

S. Villa et al. / Il Farmaco 58 (2003) 929ꢀ
/937
935
methyl-3-ammonium-1-propanesulfonate (TBSꢂ
/
0.1%
SB3-14) was added to the supernatant in 1/3 v/v
proportion. After centrifugation (55,000 rpm, 4 8C, 2
h) in a TL100 ultracentrifuge (Beckman), the PrPres
*
/
containing pellet was re-suspended in TBS (10% NaClꢂ
/
0.1% SB3-14) by sonication and re-centrifuged in a
sucrose cushion under the same conditions.
The final pellet was re-suspended by sonication in
TBS 0.1% SB3-14 and stored at ꢃ20 8C.
/
4.2.2. PrPres treatment with CR and its derivatives
Samples containing equal amounts of PrPres were
incubated at 37 8C for 1 h in the absence or presence of
CR or its derivatives, at increasing concentration (25,
50, 250, 500 and 750 mg/ml final).
After incubation, all samples were treated with
proteinase K (PK, Boehring Manheim) (300 mg/ml final
concentration, 37 8C, 1 h) and digestion was stopped by
the addition of PMSF (phenylmethylsulfonyl fluoride,
Sigma), 1 mM final concentration. Half of each PK-
digested incubation product was treated with 3 M
guanidine thiocyanate (pH 2.5) for 10 min and bovine
serum albumin (BSA) (200 mg/ml final concentration)
Scheme 5.
Fig. 1. Western blot analysis: samples containing equal amounts of PrPres were incubated at 37 8C for 1 h alone (Ctr) or with increasing
concentrations of Congo red and its derivatives. All samples were evaluated both after digestion with PK, and after treatment with 3 M guanidine
thiocyanate (GNDꢂ
/
). Above each image the name and concentration of tested compound are reported. The result of 1a was not significant because
the compound was not stable in incubation medium.

936
S. Villa et al. / Il Farmaco 58 (2003) 929ꢀ937
/
Table 2
Microanalyses
nic acid) was comparable to RC in its ability to bind
PrPres aggregates. This result seems to support the
hypothesis of an in vivo hydrogenolysis of RC by
azoreductase to give 1a as the active moiety, similarly
to other pro-drugs (e.g. sulphasalazine). It is to be noted
that we were unable to test 1b as its sodium salt 1a,
having found that it decomposed when kept in the
incubation medium at room temperature. Substitution
Formula mol. wt.
Calculated (%)
Found (%)
1a
1b
1c
1d
1f
C
260.25
10H9N2NaO3S
C 46.15
H 3.49
C 46.42
H 3.51
N 10.76
S 12.32
C 50.41
H 4.23
N 10.43
S 12.26
C 50.39
H 4.22
C10H10N2O3S
238.26
of the SO3H group of 1b with Ã/COOH (1c) or SO2NH2
N 11.76
S 13.46
C 55.36
H 4.65
N 11.71
S 13.47
C 55.28
H 4.63
(1d) retained a significant activity, though lower than
that of the parent compound 1b. Unexpectedly, removal
of the sulphonic group as in 1,2-diamino-naphthalene
(1f) led to a still active compound. Interestingly,
reduction of the unsubstituted benzenic group of 1f to
2f did not affect its activity. On the contrary, the lack of
the 2 amino group as in 1e led to an inactive compound.
The importance of this group in eliciting binding
properties to PrPres, is also supported by the inactivity
of the 4-amino-5,6,7,8-tetrahydro-napthalen-1-sulpho-
nic acid (2g).
C11H11ClN2O2
238.05
N 11.74
Cl 14.85
C 50.62
H 4.67
N 11.64
Cl 14.90
C 50.56
H 4.62
C10H11N3O2S
237.28
N 17.71
S 13.51
C 51.97
H 5.23
N 17.81
S 13.41
C 51.86%
H 5.25
C10H12Cl2N2
231.12
N 12.12
Cl 30.68
C 51.08
H 6.86
N 12.14
Cl 30.72
C 51.10
H 6.90
2f
C10H16Cl2N2
235.15
N 11.91
Cl 30.15
C 45.54
H 5.35
N 11.93
Cl 30.17
C 45.56
H 5.38
Acknowledgements
2g
C10H14ClNO3S
263.04
This research was supported by a grant from the
Italian Ministry of University and Scientific and Tech-
nological Research (Cofin, 1998).
N 5.31
Cl 13.44
S 12.16
N 5.24
Cl 13.43
S 12.25
was added; these last samples were then precipitated
with 10 volumes of methanol.
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